The Role of Ion Binding Sites in C-Type Inactivation of A K Channel

Abstract

The selectivity filter of K+ channels contains four sequential ion binding sites and serves the dual functions of selecting for K+ and acting as a gate. The process of gating at the selectivity filter is referred to as C-type or slow inactivation. During slow inactivation the selectivity filter changes conformation from a conductive to a non-conductive state. It has been well established that ion binding to the selectivity filter modulates C-type inactivation but the mechanism is not known. The major challenge in investigating the ion binding sites is that they are constructed from the peptide backbone and are therefore not accessible to modification by conventional mutagenesis. To manipulate these sites, we use chemical synthesis to introduce amide to ester substitutions in the protein backbone of the selectivity filter. For our investigation, we use the bacterial channel KcsA that is used as a model to investigate C-type inactivation. In combination with chemical synthesis, we use electrophysiological measurements to determine the effect of the ester substitution on inactivation and x-ray crystallography to determine the effect of ester substitutions on the structure and ion occupancy of the selectivity filter. Our studies reveal surprisingly distinct roles for the ion binding sites in the inactivation process. The specific roles of the ion binding sites provides insights into the structural changes at the selectivity filter that take place during C-type inactivation. This study underscores the utility of unnatural mutagenesis in investigating ion channel mechanisms.